Method and systems for processing polynucleotides

• US 10,253,364 B2
• Filed: 05/15/2018
• Issued: 04/09/2019
• Est. Priority Date: 12/14/2012
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Hindson, Christopher, Schnall-Levin, Michael, Ness, Kevin, Jarosz, Mima, Saxonov, Serge, Hardenbol, Paul, Bharadwaj, Rajiv, Zheng, Xinying, Belgrader, Phillip
• Primary Examiner(s): Thomas, David C
• Application Number:
  US15/980,473
• Publication Number:
  US 20180258482A1
• Time in Patent Office: 329 Days
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

Claims

1. A method for generating a sequencing set, comprising:

(a) bringing an aqueous fluid comprising a plurality of cells and a plurality of beads in contact with a continuous phase comprising an oil that is immiscible with said aqueous fluid, to generate a plurality droplets, wherein a droplet of said plurality of droplets comprises a single cell from said plurality of cells and a single bead from said plurality of beads, wherein said single bead comprises a plurality of nucleic acid barcode molecules, and wherein nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules comprise a common barcode sequence;

(b) in said droplet, subjecting messenger ribonucleic acid (mRNA) molecules from said single cell to reverse transcription in the presence of said nucleic acid barcode molecules to yield barcoded complementary deoxyribonucleic acid (cDNA) molecules comprising said common barcode sequence, or a complement thereof;

(c) breaking or disputing said plurality of droplets, thereby releasing a plurality of cDNA molecules from said plurality of droplets, wherein said plurality of cDNA molecules comprises said barcoded cDNA molecules or derivatives thereof; and

(d) subjecting said plurality of cDNA molecules or derivatives thereof to one or more reactions to generate said sequencing set,

wherein, after (a), said nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules are released from said single bead.

2. The method of claim 1, wherein said nucleic acid barcode molecules comprise at least 1,000 nucleic acid barcode molecules.

3. The method of claim 1, wherein said nucleic acid barcode molecules comprise at least 100,000 nucleic acid barcode molecules.

4. The method of claim 1, wherein said plurality of droplets is generated at a flow rate of at least about 1,000 Hz.

5. The method of claim 1, wherein said plurality of beads comprises a plurality of nucleic acid barcode molecules comprising barcode sequences that are different across said plurality of beads.

6. The method of claim 1, further comprising digesting remaining nucleic acid barcode molecules from said droplet subsequent to breaking or disrupting said plurality of droplets.

7. The method of claim 1, wherein said single cell and said single bead are partitioned in said plurality of droplets using a channel structure comprising a first channel in fluid communication with a first source comprising a cell suspension of said plurality of cells and a second channel in fluid communication with a second source comprising a bead suspension of said plurality of beads.

8. The method of claim 1, wherein said plurality of beads is a plurality of gel beads.

9. The method of claim 1, wherein said one or more reactions comprise one or more nucleic acid amplification reactions that generate amplified products from said plurality of barcoded cDNA molecules.

10. The method of claim 9, further comprising purifying said amplified products.

11. The method of claim 9, further comprising ligating functional sequences to said amplified products, wherein said functional sequences permit attachment of said sequencing set to a flow cell of a sequencer for nucleic acid sequencing.

12. The method of claim 9, wherein said one or more nucleic acid amplification reactions comprise a polymerase chain reaction.

13. The method of claim 1, wherein said one or more reactions comprise addition of functional sequences to said plurality of barcoded cDNA molecules, wherein said functional sequences permit attachment of said sequencing set to a flow cell of a sequencer for nucleic acid sequencing.

14. The method of claim 1, further comprising using a sequencer to subject said sequencing set to nucleic acid sequencing to generate a plurality of sequences comprising sequences corresponding to said mRNA molecules and said common barcode sequence.

15. The method of claim 1, wherein said plurality of beads have substantially monodisperse cross-sectional dimensions.

16. The method of claim 1, wherein said nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules comprise a functional sequence that facilitates sequencing of said sequencing set, and wherein said barcoded cDNA molecules comprises said functional sequence.

17. The method of claim 1, wherein said plurality of droplets comprises a transposase.

26. The method of claim 9, wherein said one or more nucleic acid amplification reactions comprise use of a primer comprising a functional sequence configured to attach to a flow cell of a sequencer, and wherein said amplified products comprise said functional sequence.

27. The method of claim 7, wherein said single cell and said single bead are partitioned in a microfluidic device comprising said channel structure.

28. The method of claim 1, wherein a droplet of said plurality of droplets comprises a nuclease.

29. The method of claim 28, wherein said nuclease is an endonuclease.

30. The method of claim 28, wherein said nuclease is a restriction endonuclease.

31. The method of claim 1, wherein, prior to (b), said single cell is lysed in said droplet.

32. The method of claim 31, wherein a droplet of said plurality of droplets comprises cell lysis reagents.

33. The method of claim 1, wherein said plurality of nucleic acid barcode molecules is releasable from said single bead upon application of a stimulus.

34. The method of claim 33, wherein said stimulus is selected from the group consisting of a photo-stimulus, a thermal stimulus, and a chemical stimulus.

35. The method of claim 33, wherein said stimulus is a chemical stimulus and wherein said plurality of droplets comprises said chemical stimulus.

36. The method of claim 35, wherein said stimulus is a reducing agent.

37. The method of claim 1, wherein said nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules comprise a bond that is cleavable upon application of a stimulus, thereby releasing said nucleic acid barcode molecules from said single bead.

38. The method of claim 37, wherein said stimulus is selected from the group consisting of a photo-stimulus, a thermal stimulus, and a chemical stimulus.

39. The method of claim 37, wherein said stimulus is a chemical stimulus, and wherein said droplet comprises said chemical stimulus.

40. The method of claim 39, wherein said chemical stimulus is a reducing agent.

41. The method of claim 37, wherein said bond is a disulfide bond.

42. The method of claim 1, wherein a subset of said plurality of droplets does not include a cell.

43. The method of claim 1, wherein a subset of said plurality of droplets does not include a bead.

44. The method of claim 1, wherein said nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules comprise a priming sequence.

45. The method of claim 16, wherein said functional sequence is a flow cell attachment sequence or a sequencing primer sequence.

46. The method of claim 1, wherein a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprise a sequence complementary to a sequence of said mRNA molecules.

47. The method of claim 1, wherein a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a poly-thymine sequence.

78. The method of claim 1, wherein in (b), said barcoded cDNA molecules are single-stranded cDNA molecules and wherein (d) comprises synthesizing double-stranded barcoded cDNA molecules.

80. The method of claim 1, wherein (d) comprises fragmenting said barcoded cDNA molecules or derivatives thereof.

82. The method of claim 1, wherein said nucleic acid barcode molecules comprise 1,000,000 nucleic acid barcode molecules.

83. The method of claim 1, wherein said nucleic acid barcode molecules comprise 10,000,000 nucleic acid barcode molecules.

84. The method of claim 1, wherein said nucleic acid barcode molecules comprise 100,000,000 nucleic acid barcode molecules.

88. The method of claim 1, wherein said plurality of droplets is generated using a microfluidic device comprising a channel structure comprising a first channel and a second channel, wherein said first channel is configured to supply a first fluid source comprising a cell suspension of said plurality of cells to said channel structure, and wherein said second channel is configured to supply a second fluid source comprising a bead suspension of said plurality of beads to said channel structure.

89. The method of claim 88, wherein said channel structure further comprises a third channel configured to supply said continuous phase to said channel structure.

92. The method of claim 1, wherein said continuous phase comprises a fluorinated oil.

93. The method of claim 92, wherein said continuous phase further comprises a fluorosurfactant.

94. The method of claim 93, wherein said fluorosurfactant inhibits coalescence of said plurality of droplets.

98. The method of claim 1, wherein said sequencing set is generated using a transposase.

100. The method of claim 1, wherein said plurality of nucleic acid barcode molecules comprise a uracil.

102. The method of claim 33, wherein said stimulus is a biological stimulus and wherein said plurality of droplets comprise said biological stimulus.

103. The method of claim 102, wherein said biological stimulus is an enzymatic stimulus.

104. The method of claim 37, wherein said stimulus is a biological stimulus and wherein said plurality of droplets comprise said biological stimulus.

105. The method of claim 104, wherein said biological stimulus is an enzymatic stimulus.

18. A method for generating a sequencing set, comprising:

(a) bringing an aqueous fluid comprising a plurality of cells and a plurality of gel beads in contact with a continuous phase comprising an oil that is immiscible with said aqueous fluid, to generate a plurality droplets, wherein a droplet of said plurality of droplets comprises a single cell from said plurality of cells and a single gel bead from said plurality of gel beads, wherein said single gel bead comprises a plurality of nucleic acid barcode molecules, and wherein nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules comprise a common barcode sequence;

(b) in said droplet, subjecting messenger ribonucleic acid (mRNA) molecules from said single cell to reverse transcription in the presence of said nucleic acid barcode molecules to yield barcoded complementary deoxyribonucleic acid (cDNA) molecules comprising said common barcode sequence, or a complement thereof;

(c) breaking or disrupting said plurality of droplets, thereby releasing a plurality of cDNA molecules from said plurality of droplets, wherein said plurality of cDNA molecules comprises said barcoded cDNA molecules or derivatives thereof; and

(d) subjecting said plurality of cDNA molecules or derivatives thereof to one or more reactions to generate said sequencing set.

19. The method of claim 18, wherein said nucleic acid barcode molecules comprise 1,000 nucleic acid molecules.

20. The method of claim 18, wherein said plurality of gel beads comprises a plurality of nucleic acid barcode molecules comprising barcode sequences that are different across said plurality of gel beads.

21. The method of claim 18, wherein said nucleic acid barcode molecules are released from said single gel bead upon exposure to a chemical stimulus in said droplet.

22. The method of claim 18, wherein said one or more reactions comprise one or more nucleic acid amplification reactions that generate amplified products from said plurality of barcoded cDNA molecules.

23. The method of claim 18, wherein said one or more reactions comprise addition of functional sequences to said plurality of barcoded cDNA molecules, wherein said functional sequences permit attachment of said sequencing set to a flow cell of a sequencer for nucleic acid sequencing.

24. The method of claim 18, further comprising using a sequencer to subject said sequencing set to nucleic acid sequencing to generate a plurality of sequences comprising sequences corresponding to said mRNA molecules and said common barcode sequence.

25. The method of claim 18, wherein said plurality of droplets comprises a transposase.

48. The method of claim 18, wherein said nucleic acid barcode molecules comprise 100,000 nucleic acid barcode molecules.

49. The method of claim 18, wherein said single cell and said single gel bead are partitioned in said plurality of droplets using a microfluidic device comprising a channel structure comprising a first channel in fluid communication with a first source comprising a cell suspension of said plurality of cells and a second channel in fluid communication with a second source comprising a bead suspension of said plurality of gel beads.

50. The method of claim 22, further comprising purifying said amplified products.

51. The method of claim 50, further comprising ligating functional sequences to said amplified products, wherein said functional sequences permit attachment of said sequencing set to a flow cell of a sequencer for nucleic acid sequencing.

52. The method of claim 18, further comprising using a sequencer to subject said sequencing set to nucleic acid sequencing to generate a plurality of sequences comprising sequences corresponding to said mRNA molecules and said common barcode sequence.

53. The method of claim 18, wherein said nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules comprise a functional sequence that facilitates sequencing of said sequencing set, and wherein said barcoded cDNA molecules comprise said functional sequence.

54. The method of claim 53, wherein said functional sequence is a flow cell attachment sequence or a sequencing primer sequence.

55. The method of claim 18, wherein said plurality of gel beads are a plurality of degradable gel beads.

56. The method of claim 55, wherein said plurality of gel beads are degradable upon application of a stimulus.

57. The method of claim 56, wherein said stimulus is selected from the group consisting of a photo-stimulus, a thermal stimulus, and a chemical stimulus.

58. The method of claim 56, wherein said stimulus is a chemical stimulus, and wherein said plurality of droplets comprises said chemical stimulus.

59. The method of claim 58, wherein said stimulus is a reducing agent.

60. The method of claim 58, wherein said single gel bead comprises a plurality of polymers and wherein said chemical stimulus depolymerizes said single gel bead, thereby degrading said single gel bead.

61. The method of claim 58, wherein said single gel bead comprises a plurality of cross-links and wherein said chemical stimulus disrupts said cross-links, thereby degrading said single gel bead.

62. The method of claim 18, wherein said nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules comprise a bond that is cleavable upon application of a stimulus, thereby releasing said nucleic acid barcode molecules from said single gel bead.

63. The method of claim 62, wherein said stimulus is selected from the group consisting of a photo-stimulus, a thermal stimulus, and a chemical stimulus.

64. The method of claim 62, wherein said stimulus is a chemical stimulus and wherein said plurality of droplets comprises said chemical stimulus.

65. The method of claim 64, wherein said chemical stimulus is a reducing agent.

66. The method of claim 62, wherein said bond is a disulfide bond.

67. The method of claim 22, wherein said nucleic acid amplification reaction comprises use of a primer comprising a functional sequence configured to attach to a flow cell of a sequencer, and wherein said amplified products comprise said functional sequence.

68. The method of claim 18, wherein a droplet of said plurality of droplets comprises a nuclease.

69. The method of claim 68, wherein said nuclease is an endonuclease.

70. The method of claim 68, wherein said nuclease is a restriction endonuclease.

71. The method of claim 18, wherein, prior to (b), said single cell is lysed in said droplet.

72. The method of claim 71, wherein a droplet of said plurality of droplets comprises cell lysis reagents.

73. The method of claim 18, wherein a subset of said plurality of droplets does not include a cell.

74. The method of claim 18, wherein a subset of said plurality of droplets does not include a gel bead.

75. The method of claim 18, wherein said nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules comprise a priming sequence.

76. The method of claim 18, wherein said nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules comprise a sequence complementary to a sequence of said mRNA molecules.

77. The method of claim 18, wherein said nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules comprise a poly-thymine sequence.

79. The method of claim 18, wherein in (b), said barcoded cDNA molecules are single-stranded cDNA molecules and wherein (d) comprises synthesizing double-stranded barcoded cDNA molecules.

81. The method of claim 18, wherein (d) comprises fragmenting said barcoded cDNA molecules or derivatives thereof.

85. The method of claim 18, wherein said nucleic acid barcode molecules comprise 1,000,000 nucleic acid barcode molecules.

86. The method of claim 18, wherein said nucleic acid barcode molecules comprise 10,000,000 nucleic acid barcode molecules.

87. The method of claim 18, wherein said nucleic acid barcode molecules comprise 100,000,000 nucleic acid barcode molecules.

90. The method of claim 18, wherein said plurality of droplets is generated using a microfluidic device comprising a channel structure comprising a first channel and a second channel, wherein said first channel is configured to supply a first fluid source comprising a cell suspension of said plurality of cells to said channel structure, and wherein said second channel is configured to supply a second fluid source comprising a bead suspension of said plurality of beads to said channel structure.

91. The method of claim 90, wherein said channel structure further comprises a third channel configured to supply said continuous phase to said channel structure.

95. The method of claim 18, wherein said continuous phase comprises a fluorinated oil.

96. The method of claim 95, wherein said continuous phase further comprises a fluorosurfactant.

97. The method of claim 96, wherein said fluorosurfactant inhibits coalescence of said plurality of droplets.

99. The method of claim 18, wherein said sequencing set is generated using a transposase.

101. The method of claim 18, wherein said nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules comprise uracil.

106. The method of claim 57, wherein said stimulus is a biological stimulus and wherein said plurality of droplets comprise said biological stimulus.

107. The method of claim 106, wherein said biological stimulus is an enzymatic stimulus.

108. The method of claim 62, wherein said stimulus is a biological stimulus and wherein said plurality of droplets comprise said biological stimulus.

109. The method of claim 108, wherein said biological stimulus is an enzymatic stimulus.